Low FasL levels promote proliferation of human bone marrow-derived mesenchymal stem cells, higher levels inhibit their differentiation into adipocytes.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into several cell types. Bone marrow (BM)-MSCs mainly differentiate into osteoblasts or adipocytes. MSC interactions with their microenvironment directly affect their self-renewal/differentiation program. Here, we show for the first time that Fas ligand (FasL), a well-explored proapoptotic cytokine, can promote proliferation of BM-derived MSCs in vitro and inhibits their differentiation into adipocytes. BM-MSCs treated with a low FasL dose (0.5 ng/ml) proliferated more rapidly than untreated cells without undergoing spontaneous differentiation or apoptosis, whereas higher doses (25 ng/ml) induced significant though not massive BM-MSC death, with surviving cells maintaining a stem cell phenotype. At the molecular level, 0.5 ng/ml FasL induced ERK1/2 phosphorylation and survivin upregulation, whereas 25 ng/ml FasL induced caspase activation. Importantly, 25 ng/ml FasL reversibly prevented BM-MSC differentiation into adipocytes by modulating peroxisome proliferator-activated receptor gamma (PPARγ) and FABP4/aP2 expression induced by adipogenic medium. All such effects were inhibited by anti-Fas neutralizing antibody. The in vitro data regarding adipogenesis were confirmed using Fas(lpr) mutant mice, where higher PPARγ and FABP4/aP2 mRNA and protein levels were documented in whole tibia. These data show for the first time that the FasL/Fas system can have a role in BM-MSC biology via regulation of both proliferation and adipogenesis, and may have clinical relevance because circulating Fas/FasL levels decline with age and several age-related conditions, including osteoporosis, are characterized by adipocyte accumulation in BM.

Microcirculation in rat soleus muscle after eccentric exercise: the effect of nifedipine.

This paper explores the role of the calcium entry blocker nifedipine in the explanation of eccentric exercise-induced fibre damage by changes in skeletal muscle microcirculation. Eccentric exercise (EE) was induced by indirect stimulation of rat soleus muscle in its lengthening phase during cycling. Muscle damage was assessed by histology, electron microscopy and muscle tension 48 h later. Diameters of arterioles and venules, their response to dilator and constrictor stimuli and pattern of capillary flow were measured in epiiluminated muscles using intravital microscopy. Tetanic tension developed by EE muscles was lower (8.60 +/- 1.02, means +/- SEM, n = 8 N g(-1) wet weight compared to 12.25 +/- 0.56 in controls, P < 0.01). Electron microscopy showed changes similar to those in muscles exposed to EE by downhill running (Z line streaming, disruption of sarcolemma, swollen tubules). A total of 16% of muscle fibres were damaged, and fibre areas and interstitial space were enlarged. Capillary red blood cell flow showed tendency to a greater intermittency. Large venules were narrower, but arterioles and smaller venules had diameters similar to control muscles. Vessel dilatation to topically applied 10(-4) M adenosine was attenuated. Daily administration of calcium entry blocker nifedipine by gavage (2 mg/kg/day in two equal doses) removed the narrowing of venules, restored the dilator response of all vessels to adenosine and increased capillary:fibre ratio. The percentage of damaged fibres decreased to 4.7 and the size of the interstitial space and fibre areas was normalized. Thus muscle damage caused by eccentric exercise was attenuated by nifedipine due to its beneficial effect on muscle microcirculation, which was impaired by eccentric exercise.

Determination of high-energy phosphate compounds and inorganic phosphate by reversed-phase high-performance liquid chromatography: evaluation of myocardial metabolic status in aerobically perfused and hypoxic mouse heart.

The present paper describes a simple HPLC method designed for measuring high-energy phosphate (HEP) compounds in a single run and inorganic phosphate (Pi) in an other short run under the same HPLC conditions. Inorganic phosphate was estimated by using thymidine phosphorylase (EC 2.4.2.4) which catalyzes a reaction involving inorganic phosphate to produce 2-deoxyribose 1-phosphate and thymine. The thymine/Pi stoichiometry was 1. The method provides a reproducible instrument for evaluating myocardial high-energy metabolism under physiological and pathological conditions.

Reversible binding of glycolytic enzymes and size change in the actin-containing filaments of the frog skeletal muscle.

Electron microscopy was used in order to study the change of the actin-containing filaments when the bound glycolytic enzymes were removed by a high ionic strength solution. The effectiveness of the extraction medium was checked by estimating the aldolase activity released. Evidence was provided that the larger diameter of the actin filaments in the I-bands in comparison with the A-bands can be accounted for by the bound glycolytic enzymes.

Rat motor neuron plasticity induced by dorsal rhizotomy.

The control of peripheral structural plasticity of motor neurons by primary sensory neurons was studied in rat extensor digitorum longus (EDL) muscle. Polyinnervation of muscle fibers, sprouting and the motor neuron peripheral field size following L4 dorsal root cutting were evaluated using three different approaches: intracellular recording of end plate potentials, histochemical demonstration of sprouting and polyinnervation and in vivo recording of nerve-evoked twitch. Nodal sprouting was found in rhizotomized rats but not in controls and consistently muscle polyinnervation appeared. The muscle portion innervated by L3 ventral root was relatively reduced and that innervated by L5 was relatively enlarged: a trend to caudal shift of muscle innervation arose in rhizotomized rats. A control of motor neuron plasticity by primary sensory neurons is suggested.

Gamma irradiation can reduce muscle damage in mdx dystrophic mice.

We report the effects of a single gamma irradiation delivered to the soleus muscle of one limb of normal and mdx mice at the age of 16-20 days. At 45, 75 and 90 days of age transverse cryostat sections from the mid-belly of the muscles were used for microscopic examination. In normal mice the growth of fibres was appreciably reduced by irradiation without fibre loss. In the irradiated soleus of mdx mice the number of the regenerated centrally nucleated fibres was very small and the total number of fibres was remarkably reduced. The number of the peripherally nucleated fibres, presumably surviving since the birth of the animal, was almost consistently larger than in the contralateral non-irradiated limb. The cross-sectional area of the irradiated fibres was smaller. It is well known that proliferation and fusion of satellite cells are required both for regeneration after fibre damage and for the normal postnatal growth of muscle fibres: irradiation appears to reduce regeneration and growth. It is suggested that irradiation reduces damage by reducing fusion associated with growth. Our hypothesis indirectly indicates a significant link between dystrophin deficiency and fibre necrosis and accounts well for many features of mouse dystrophy under natural and experimental conditions.

The effect of alpha 1 adrenoceptor antagonist prazosin on capillary supply, blood flow and performance in a rat model of chronic muscle ischaemia.

OBJECTIVE: This study explores whether the a1 blocker prazosin improves capillarisation, blood flow and muscle performance in chronically ischaemic muscles. DESIGN: Rat skeletal muscles were made ischaemic by unilateral ligation of the common iliac artery. The animals received prazosin and muscle blood flow, performance and capillary supply were studied 2 or 5 weeks later. MATERIALS AND METHODS: Prazosin (50 mg/l) was given in drinking water to animals with limited or intact blood supply. Muscle isometric twitch tension and fatigue index and blood flow at rest and during contractions (estimated by radio labelled microspheres) were measured in tibialis anterior, TA, and extensor digitorum longus, EDL. Capillary supply (capillary/fibre ratio, C/F) was estimated in frozen muscle sections stained for alkaline phosphatase in TA, EDL and soleus, S. RESULTS: Prazosin increased significantly capillary supply in ischaemic muscles after 2 and 5 weeks and blood flow during contractions (control muscles 163 +/- 6 ml. 100 g-1 min-1; ligated 2 weeks 25 +/- 7, ligated +prazosin 116 +/- 38 ; ligated 5 weeks 27 +/- 7, +prazosin 137 +/- 29, p < 0.05 vs. ligated). Fatigue index (final/peak tension over 5 min contractions at 4 Hz in %) was significantly improved by prazosin after 2 weeks (FI = 69 +/- 3% controls, 35 +/- 4% ligation, 60 +/- 3% ligation +prazosin) and less after 5 weeks (52 +/- 12% ligation, 61 +/- 3% ligation +prazosin). In contrast, prazosin had no effect on muscle performance in normal muscles in spite of it increased C/F and blood flow. CONCLUSIONS: Alpha1 adrenoceptor blockade is an effective way for improvement of capillary supply, blood flow and muscle performance in ischaemic muscles; the effect on muscle performance is most pronounced 2 weeks after induction of ischaemia.

Location of the binding site of the mannose-specific lectin comitin on F-actin.

We have used electron microscopy and computer image processing to produce a three-dimensional reconstruction of F-actin filaments decorated with the putative lectin and actin-binding protein comitin. These reconstructions show that comitin binds to F-actin at high radius primarily to actin subdomain 1. This location is distinctly different from the binding site on F-actin for other actin bundling proteins, such as members of the alpha-actinin family, and may result from the positively charged comitin interacting with negatively charged sites near the actin N terminus in subdomain 1. The location of the comitin binding site and its restriction to subdomain 1 on a single actin monomer is consistent with comitin's having a function distinct from other actin-binding proteins and, for example, would enable comitin to link bundled actin filaments to the Golgi.

Postsynaptic effects of methoctramine at the mouse neuromuscular junction.

Functional studies were performed to evaluate the effects of methoctramine at the neuromuscular junction of the mouse. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analysed by means of the extracellular recording with an EPC7 Patch Clamp amplifier. This electrophysiological method revealed a dose-related inhibitory effect of methoctramine on the studied parameters. The dramatic reduction of the kinetics of the quantal conductance change indicates an action at the postsynaptic level. The effects of methoctramine have been compared with those of the muscarinic agonist oxotremorine. Concentration/response curves for the two drugs were obtained and the apparent EC50 values calculated. The effects of oxotremorine were not antagonized by 1 microM methoctramine. These findings suggest an interaction of some muscarinic agents on the postsynaptic receptor-ion-channel complex at the mouse neuromuscular junction.

Muscarinic modulation of neurotransmission: the effects of some agonists and antagonists.

1. Functional studies were performed to evaluate the effects of some muscarinic agents at the neuromuscular junction of the mouse. 2. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analyzed by means of extracellular recording. The amplitude of spontaneous and of evoked acetylcholine release, the frequency of spontaneous acetylcholine release and the time course of the quantal release have been measured by means of an EPC7 patch clamp amplifier. 3. This electrophysiological method revealed multiple dose-related effects of some agonists and antagonists on the above parameters. Concentration-response curves related to the parameters underlying the function of this cholinergic synapse were obtained and the apparent EC50 values calculated. 4. Many of the interactions of the agonists and antagonists could inhibit neuromuscular transmission. The rank order potencies related to the inhibition of the evoked signals were carbachol > oxotremorine > d,l-muscarine for the agonists and methoctramine > 4-DAMP > l-hyoscyamine > AFDX-116 > ipratropium > pirenzepine for the antagonists. 5. These findings suggest a more complicated pattern related to the muscarinic action at the mouse neuromuscular junction with the involvement of some post-synaptic located sites.

[Presynaptic effects induced by pretreatment with formamide of the neuromuscular junction of the mouse]. / Effetti presinaptici indotti dal pretrattamento con formamide sulla guinzione neuromuscolare di topo.

Some techniques to block muscular nerve evoked contraction involve pharmacological approaches using synaptic blocking agents. Such methods interfere with normal synaptic transmission, and could introduce artifacts making difficult the experimental interpretation. The method based on the use of formamide pre-treatment should not interfere with synaptic physiology, indeed previous works suggest that the mechanism involved in block of muscle activity could depend on the decrease in specific postsynaptic membrane capacitance, and on the disruption of the morphology of the transverse tubule system. To prove this assumption we evaluated before and after formamide pre-treatment, some pre and postsynaptic parameters related to the spontaneous quantal release (MEPC). By means of the Loose patch clamp technique, we demonstrated, that formamide pre-treatment increases in an irreversible manner the frequency of spontaneous quantal release. Morphology of MEPC appear not modified by formamide pretreatment, which does not interfere with postsynaptic cholinergic receptors activity.

[Analysis of miniature potentials in reinnervated rat muscle]. / Analisi dei potenziali in miniatura nel muscolo reinnervato di ratto.

Miniature endplate potentials (MEPPs) are regarded as the expression of release of a single quantum of acetylcholine by motor nerve endings in the muscle. Mepp frequency is dependent on the presynaptic mechanism, but MEPP amplitudes and time courses are the result of the characteristics of pre- and postsynaptic structures and of the interaction between them. After post-traumatic reinnervation of skeletal muscles, MEPP frequency increases, reaching slowly normal values. Two groups of male, Sprague Dawley rats were used: in the first group left sciatic nerve was crushed and nerve fibres were allowed to regenerate, whereas the others were regarded as controls. MEPPs were intracellularly recorded in end plates of normal and reinnervated left extensor digitorum longus muscle. MEPPs were sampled and recorded on a personal computer, and, subsequently, amplitude, rise time and half decay time were computed. At early stage after reinnervation, MEPPs showed rise times and decay times longer than normal. Afterwards, we did not find differences between mepp time courses by normal and reinnervated end plates. The possible relationships between the results and changes in acetylcholine receptor number and type, and in acetylcholinesterase activity occurring during denervation and reinnervation are discussed.